Cong Nghe Sinh Hoc Trong Xu Ly Chat Thai Nhiem Dau

7/31/2019 Cong Nghe Sinh Hoc Trong Xu Ly Chat Thai Nhiem Dau

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M U

Cng nghip du kh l mt trong nhng ngnh cng nghip quan trng,

em li li ch v cng ln cho nn kinh t quc dn. Ngun nguyn liu du

kh to ra rt nhiu sn phm phc v cho hu ht cc lnh vc cng nghip,nng nghip, i sng x hi, an ninh quc phng Bt k mt nc no s

hu ngun ti nguyn qu gi ny u c tim nng pht trin kinh t vt tri,

v vy n khng ngng c tm kim, khai thc v vi quy m ngy cng tng.

c bit vi mt nc ang pht trin nh nc ta th vai tr ca n li cng

quan trng.

Hin Vit Nam l nc khai thc du ng th 3 ng Nam , tn du u

tin c khai thc vo nm 1986 ti Vng Tu, tnh n nm 2004 khai thcc tn du th 140 triu tn, hng nm em li mt phn ba ngun ngn sch

quc gia. Hot ng thm d khai thc du kh din ra ht sc si ni thm lc

a pha Nam v ang c xu hng chuyn sang min Bc nh Thi Bnh, Hi

Phng.

Bn cnh nhng mt tch cc , hot ng khai thc cng mnh m cng

ng ngha vi vic thi ra mi trng cng nhiu ph thi v tnh trng nhim

li trm trng thm. Trong ngnh cng nghip du kh, mn khoan l mt nguncht thi v cng ln v c nguy c gy nhim rt cao, c bit l khi s dng

cc dung dch khoan gc du. N c to ra khi tin hnh khoan thm d v

pht trin m, gm hn hp t cng vi cc ho cht trong dung dch khoan

ln vo. Khi qu trnh khoan s dng dung dch gc du th tnh trng nhim

cng ln. V nguyn tc ngun ph thi ny phi c em vo b x l, tuy

nhin vic x l chng rt kh khn v tn km nn cc nh thu thng sao

lng. Hin ti trong ngnh cng nghip du kh nc ta ch mi p dng ccbin php ho l thng thng lm gim tc hi, m khng x l trit ri

thi vo mi trng.

Vn t ra cho cc nh mi trng l phi x l chng, phng n gii

quyt phi m bo sao cho va hiu qu, n gin m li kinh t. Do m

phng php sinh hc ang c ch bi cc u im vt tri ca n so vi

cc phng php ho l nh: an ton vi mi trng, n gin, x l trit m

gi thnh li r. Mt trong cc phng php x l sinh hc em li hiu qu cao l s dng cc cht hot ho b mt do cc vi sinh vt to ra. Cht hot ho

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b mt sinh hc l mt hp cht lng cc cho php ho tan cc cht khng tan

vo trong nc, chng d b phn hu sinh hc li khng c thm vo n c

th sn xut t cc ngun c cht ph thi ca cc ngnh cng nghip khc gi

thnh h li gii quyt c tnh trng nhim do cc ngun ph thi ny sinh

ra. x l mn khoan, CHHBM t ra rt c trin vng. Vit Nam, mi ch c mt s t cc cng trnh nghin cu v cht hot

ho b mt sinh hc, hng nghin cu v ng dng chng cn rt mi m.

Xut pht t nhng yu cu thc t t ra, chng ti tin hnh nghin cu

ti: nh gi kh nng to CHHBMSH do cc chng vi khun phn lp

t mn khoan du kh Vng Tu v nh hng ca chng ln kh nng

phn hu du.

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CHNG 1. TNG QUAN TI LIU

1. TNH HNH S DNG DUNG DCH KHOAN V VN X L MN

KHOAN DU KH HIN NAY

1.1. Tnh hnh s dng dung dch khoanTrong ngnh cng nghip khai thc du kh, khi tin hnh khoan m bi

trn mi khoan, vn chuyn mn khoan t y ging ln trn b mt, gi p sut

va n nh ngn hin tng phun tro, bo v thnh ging khi st l, bi

trn chng khoan, cn khoan ngi ta lun phi s dng dung dch khoan

(DDK).

DDK l mt h dung dch bao gm hn hp cc cht iu chnh nht,

cht to nh, cht phn tn, cht dit khun, cht ph gia, NaCl, CaCl 2, vi, st,BaSO4, cht chng mt nc... nhm to cc c tnh mong mun cho DDK.

Khi chng c pha vi nc gi l DDK gc nc, hoc pha vi du gi l

DDK gc du. Hm lng du gc trong DDK thng cha trn 70%.

So vi DDK gc nc th DDK gc du t v c hi hn, nhng chng

c nhng c tnh hn hn nh: n nh c nhit cao, chng st l tt, kh

nng gi dung dch tt... Khi tin hnh khoan su, khoan nghing m bo

thnh cng ngi ta lun s dung dch khoan gc du.Du gc ca cc dung dch khoan u tin thng l du diezen hay du

th. Do gy nh hng nghim trng ti mi trng m hin nay xu hng s

dng cc loi du gc c c thp nh du khong, du thc vt hay du tng

hp c c thp.

Du khong c sn xut t du th c loi b cc hp cht c hi

nh cc hp cht thm, d vng nn c thp hn du th.

Du thc vt c nht cao nn thng s dng cc dn xut ca n c

nht thp hn, u im ca n l c kh nng phn hu sinh hc tt hn du th

nn t nh hng ti mi trng hn.

Du tng hp c c thp, d phn hu sinh hc, gi thnh li r hn

du thc vt nn c s dng rng di [5].

Hin nay trn th gii v Vit Nam, mt s nh thu s dng DDK

gc du tng hp saraline c c thp. Saraline c sn xut t du khong

loi b cc hp cht thm d vng c hi, n khng cha cc hp cht thm a

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vng vn l mt hp cht rt c hi cho mi trng, thnh phn chnh ca n l

cc ankan mch thng t C12 nC25.

Tuy cc du gc c x l loi b cc thnh phn c hi, chng vn

gy tc ng ln ti mi trng sinh thi v sc kho con ngi. Cc du gc

cha cc thnh phn hydrocacbon, cc thnh phn ny nu tch t nng caos nh hng ti qu trnh trao i cht ca sinh vt. Trong mi trng nc n

to lp vng trn b mt ngn cn s ho tan oxy vo trong nc, s trao i

nhit gia kh quyn v thu quyn, khng cho nh sng i vo nc t nh

hng ti ngun thc n, dinh dng, h hp ca sinh vt, ti cc loi u

trng... i vi mi trng t, du y nc v khng kh ra ngoi lm cho t

mt kh nng hp th v trao i, ngn cn qu trnh trao i cht ca h vi sinh

vt trong t. i vi con ngi, n nh hng thng qua mi trng nc,khng kh, n i vo trong li thc n, gy tc ng ti h tiu ho, h bi tit,

h h hp, h thn kinh.

C th nh du saraline, c nhng cng b v tc hi gy bnh vim

phi ca loi du ny [21].

1.2. Vn x l mn khoan du kh hin nay

1.2.1. Mn khoan

Mn khoan l sn phm ph thi ca ngnh cng nghip du kh, gm hn

hp ca cc loi t b v vn ra khi tip xc vi mi khoan v c vn

chuyn ln trn mt t thng qua qu trnh tun hon ca dung dch khoan. H

dung dch khoan c bm vo bn trong ng khoan, nht v vn tc tun

hon duy tr mn khoan trng thi huyn ph, thng qua khng gian gia

thnh ng khoan v thnh l khoan n vn chuyn mn khoan ln trn mt, ti

n tch khi mn khoan v li tun hon tr li ging khoan. Do , ngoi

t , mn khoan cn ln cc ho cht trong DDK. i vi qu trnh s dng

DDK gc du th mn khoan thm y du.

Mn khoan c to ra t hot ng khoan thm d v pht trin m. Khi

lng mn khoan ph thuc vo ng knh ging, tc khoan v chiu di

ca mi cng on khoan.

Trong qu trnh khoan thm d, lng mn khoan to ra t, khi a ln mt

t, mn khoan c ra sch, mt phn gi li nghin cu, theo quy ch mn

khoan loi b phi a vo b chn, nc ra mn khoan phi c x l sch.

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Tuy nhin, cc quy nh ny rt kh thc hin ng thi gy tn km nn cc

nh thu thng trn trnh trch nhim.

Giai on khoan pht trin m, s lng ging khoan nhiu cng vi lng

mn khoan mi ging rt ln, khoan khai thc thng khng ly mu nn

lng mn li v cng ln. Ch tnh ring m Bch H, trung bnh khoan mtging su 4000m to ra khong 350 m3 (900 tn) mn khoan. Nu duy tr t l

du 70% trong DDK gc du th mn khoan thi ra c hm lng du v cng

ln.

1.2.2. Tc hi ca mn khoan nhim du

Vi c th l mt sn phm ph thi nhim du, c s lng rt ln, mn

khoan v ang gy nhng tc hi khng nh.

Khi thi ra ngoi bin mn khoan nhim du lm thay i mi trng sng

ca cc sinh vt y, ring cc cht c hi trong mn khoan s xm nhp vo

trong thc n, tc ng ti qu trnh trao i cht, c th git cht hoc gy bin

dng ging loi, lm thay i h sinh thi bin, nh hng nhim d lan rng

c bit l khu vc c dng hi lu.

Ti t lin vic cht ng mn khoan hay lm vt liu chn lp h, ao h

chim din tch ln, lm thay i a hnh v sau khi cn dng din tch th

phi tn km chi ph bc d, khng nhng th n cn lm nhim ngun nc,

khng kh qua nh hng ti sc kho ca con ngi [5].

1.2.3. Tnh hnh x l mn khoan nhim du

Hin nay nc ta v nhiu nc trn th gii, vic x l mn khoan ch

mi p dng cc bin php ho l nhm gim thiu lng du bm dnh trn mn

khoan trc khi thi xung bin, hoc vn chuyn vo b lp cc a im

to b mt xy dung hay x l thnh cc nguyn vt liu xy dng nh ThiLan, Malayxia, Ty u. Cc nc pht trin trnh thp th cha quan tm

ti mi trng thng chp nhn mt khon ph bi thng ri cho php tu

tin cc cht thi [5].

Nc ta hin ny cng p dng mt s cc bin php gim tc ng ca

mn khoan i vi ngun li thu hi sn v mi trng bin:

- S dng sng rung li v cc thit b li tm hiu qu cao trn gin khoan

gim thiu ti a hm lng du bm dnh trn mn khoan n di 10%.

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- Thc hin chng trnh gim st mi trng ti cc khu vc ln cn ca

ging khoan nh gi tnh trng thay i mi trng.

- Nghin cu c ca cc loi du tng hp i vi sinh vt.

Tuy nhin, cc bin php trn khng gii quyt c vn trit , mi

trng sinh thi vn b tc ng ln.

2. PHNG PHP SINH HC TRONG X L NHIM DU

2.1. Vai tr ca vi sinh vt trong x l nhim du

2.1.1. H vi sinh vt trong mn khoan du kh

Nh ni trn, mn khoan l hn hp cc loi t trong lng t

chuyn ln. Mn khoan cha mt phn cc ph gia trong dung dch khoan, phn

du (i vi DDK gc du), nc trong lng t v khu h vi sinh vt trong

ging khoan. Cc cht thng s dng trong dung dch khoan l: tinh bt,

cacbonoxymetylcellulo, lignosulfonat, cc loi gum t nhin, du nh ho, du

gc...[23]. Cc cht ny c nhiu nh khoa hc chng minh l c kh nng

b phn hu bi cc vi sinh vt:Bacillus subtilis,Pseudomonas sp., cc vi khun

kh sunphat nh Desulfovibrio desulfuricans [15]... Mc d trong mn khoan

thng b sung cc cht dit khun nhng kh nng b tn cng bi vi sinh vt

ca cc cht k trn vn rt cao. Vic c mt nc trong mn khoan cng lm

tng kh nng phn hu du ca vi sinh vt. Smirnova chng minh c nhiu

loi vi khun bao gm c cc loi oxy ho hydrocacbon c mt trong DDK [15].

Do mi vng a l c khu h vi sinh vt khc nhau nn cc mu mn

khoan ly cc a im khc nhau c khu h vi sinh vt c trng cng khc

nhau.

H vi sinh vt trong mn khoan l c s cho cc bin php x l du bng

bin php sinh hc.2.1.2. C ch phn hu cc hydrocacbon

C ch phn hu cc hydrocacbon ca vi sinh vt c cc nh khoa

hc trong v ngoi nc nghin cu. Vi sinh vt s dng hydrocacbon lm

ngun dinh dng v nng lng cho s sinh trng v pht trin. Vic s dng

cc hydrocacbon ca vi sinh vt c th xy ra theo hai hng:

- i vi mt s cc hydrocacbon tan trong nc, vi sinh vt c th hp th

trc tip.

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- Vi cc hydrocacbon kh tan m c th tan di dng nh tng du

-nc th qu trnh phn hu vi sinh theo trnh t cc bc: u tin l ho tan

cc hydrocacbon di dng nh tng du nc bng cch tit ra cc cht hot

ho b mt sinh hc, sau vi sinh vt tip xc vi du, cui cng n tit ra cc

enzyme chuyn ho cc hydrocacbon thnh cc cht m n c th s dngc.

Nhn chung cc hydrocacbon khc nhau b phn hu bi nhiu loi vi

khun v bng nhiu con ng khc nhau.

2.1.2.1. C ch phn hu cc ankan

Kh nng phn hu ca cc ankan ph thuc vo cu trc phn t ca

chng. Thng thng cc hydrocacbon bc 1 v bc 2 d phn hu hn cc

hydrocacbon bc 3 v 4.

a. C ch phn hu cc n-ankan:

Qu trnh phn hu ankan c khi mo nh enzym mono-oxygenaza v

di-oxygenaza. Qu trnh ny i hi c s tham gia ca mt phn t oxy v cht

cho in t NADPH2.

Cc giai on oxy ho ankan:

* Giai on 1: To thnh ru, xy ra qua 2 bcTo thnh hp cht peoxyt

R- CH2- CH3 + O2 > R- CH2- CH2- OOH

Hp cht peoxyt khng bn di tc dng ca NADPH2 to thnh ru v

nc:

R- CH2- CH2- OOH + NADPH2 > RCH2- CH2- OH + H2O + NADP (2)

T ankan chuyn thnh ru c hai kh nng xy ra:

To thnh ru bc 1: xy ra khi nhm OH gn vo C bc 1 (2)

To thnh ru bc hai: khi nhm OH gn vi C bc 2:

R-CH2-CH2 - OOH + O2 + NADPH2 > R- CH- CH2 + H2O + NADP

|

OH

* Giai on 2:

Ru bc 1 to thnh andehit

R- CH2 - CH2 - OH +1/2 O2 > R- CH2 - CHO + H2O

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Ru bc 2 to thnh xeton:

R - CH - CH3 + 1/2 O2 > R CH - CH3 +H2O

| ||

OH O

* Giai on 3: to thnh axit bo:

- Cc xeton b oxy ho to thnh este, lin kt este b ph v to ra mt axit

v ru bc mt, ru bc 1 li b oxy ho thnh andehit ri axit bo:

R- CH2- C- CH3 >R- CH2-O- C - CH3>R- CH2 - OH + CH3 - COOH

|| ||

O O

- Cc andehit b oxy ho thnh axit bo:

R - CH 2- CHO +1/2 O2 >R - CH2 - COOH

C trng hp s oxy ho xy ra c hai u ca ankan to thnh cc diol,

cc dicacbonxylic.

* Giai on 4: cc axit bo b oxy ho tip nh tru trnh - oxy ho.

Axit bo mch di di tc dng ca mt loi enzym chuyn sang dng

acetyl coenzym A v chuyn ho tip di tc dng ca nhiu enzym khc. Ktqu l sau mi chu k chuyn ho, mt nhm acetyl CoA b ct ra v phn t

axit bo b ct i hai nguyn t cacbon. Sn phm cui cng ca chu trnh

oxy ho l CO2 v nc.

b. C ch phn hu cc alkan mch nhnh:

Do cn tr v mt khng gian nn kh nng phn hu ca chng km hn

n-ankan. Theo Atlas, cc vi sinh vt phn hu u tin C bc1 v bc 2, cn C

bc 3 v 4 th kh phn hu hn.Ankan c nhm metyl du mch kh phn hu hn gia mch.

Ankan c mch nhnh di d b phn hu hn ankan c mch nhnh ngn.

Trong qu trnh nghin cu s phn hu cc ankan mch nhnh, ngi ta

xut bin dng ca chu trnh TCA l chu trnh Metylcitrat, thay vo v tr ca

axit citric l metylcitrat.

c. C ch phn hu cc cycloankan:

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Cycloankan l cu t chnh ca du th. Khi mo cho s phn hu

cycloankan cng l cc enzym monooxygenaza v oxygenaza. Di tc dng

ca cc enzym ny, cc cycloankan b phn hu thnh cc cycloankanol.

Theo cc tc gi, kh nng phn hu ca cyclohexan l mnh nht trong

dy ng ng cycloankan. Cng vng cycloankan cht no c mch nhnh dihn th s d phn hu hn.

Khi phn hu cyclohexan, qu trnh hydroxyl ho c xc tc bi enzym

oxydaza chc nng to ra mt ru mch vng. Ru mch vng s b dehydro

ho to ra xeton, xeton b oxy ho tip thnh lacton. Lacton s b thu phn,

nhm hydroxyl b oxy ho thnh mt nhm andehit v mt nhm cacboxyl. Kt

qu l axit dicacboxylic b bin i tip nh chu trnh - oxy ho.

Cc vi sinh vt c kh nng pht trin trn cyclohexan phi thc hin tt c

cc phn ng trn. Tuy nhin ta thng gp hn cc vi sinh vt c kh nng

chuyn cyclohexan thnh ru mch vng nhng khng c kh nng lacton ho

v m mch vng. Do vy c ch cng sinh v cng trao i cht (co-

metabolism) ng mt vai tr rt quan trng trong phn hu sinh hc cc hp

cht hydrocacbon mch vng.

2.1.2.2. C ch phn hu hydrocacbon thm

a. Benzen

Xc tc ban u cho qu trnh oxy ho benzen l enzym dioxygenaza, tc

l phn t oxy s gn trc tip vo cacbon ca vng thm.

b. Naphtalen

Xc tc u tin l enzym oxygenaza, gn phn t oxy vo phn t

naphtalen to thnh cis-1,2 dihydroxyl 1,2 dihydro naphtalen, sau tch

nc thnh 1,2-dihydroxynaphtalen. Ti y, vng b ct to thnh cis-o-hdroxyl benzan pyruvic axit, sau tip tc b oxy ho to thnh salicyandehyd,

salicylic axt v catechol, tip ct cc vng khc theo hng -octhor hay

-metha ph thuc vo chng vi khun.

Cng c trng hp xc tc oxy ho naphtanen l enzym

monooxygenaza, tc l mt nguyn t ca phn t oxy s gn vo vng

naphthalen v cui cng cho ta cis-1,2-dihydroxyl 1,2 -dihydronaphtalen. Nm

mc l nhm vi sinh vt chim u th v chng loi c kh nng phn hunaphtalen.

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Nh vy, naphtalen dn dn s b chuyn ho thnh phn t c khi lng

b hn nh vi sinh vt, lm cho du nng tr thnh du nh hn, v nht ca

du s gim i.

2.2. Cc yu t nh hng ti kh nng phn hu du ca vi sinh vt

S pht trin ca vi sinh vt phn hu du ph thuc rt ln vo cc ngunc cht v cc iu kin ca mi trng sng: nhit , pH, p sut.

Trong t nhin, s tn ti ca cc vi sinh vt c kh nng s dng

hydrocacbon rt a dng. Chng c th sng c nhng ni c iu kin rt

khc nghit nh trong ging khoan c su hng nghn mt vi p sut t 200

400 atm v nhit ln hn 1000C. Mt khc chng c th sng cc vng

ven bin, ni m hng gi c s thay i ln v mui v nhit do tc ng

ca sng v thu triu. Do s tn ti v pht trin ca vi sinh vt c kh nng

s dng hydrocacbon trong di nhit v p sut kh ln. a s cc vi sinh

vt ph hp vi nhit t 25 300C v p sut 1 atm [4].

Mt nhn t mi trng na tc ng ti s phn b v pht trin ca vi

sinh vt l pH. a s cc vi sinh vt sng mi trng c pH trung tnh hoc

kim yu.

Kh hu cng nh hng ln n s pht trin ca vi sinh vt. Nc ta c

kh hu nhit i gi ma. Ma h, nhit cao v m ln to iu kin ti

u cho vi sinh vt pht trin. Ma ng, nhit v m gim th kh nng

pht trin ca vi sinh vt cng yu i.

Bn cnh , s pht trin ca vi sinh vt ph thuc nhiu vo thnh phn

mui khong trong mi trng. S pht trin ca vi sinh vt s dng du vng

thm lc a phong ph v a dng hn i dng. Trong cc nguyn t dinh

dng khong N v P l hai nguyn t chnh nh hng ti kh nng s dng

hydrocacbon ca vi sinh vt. Ti nhiu vng nhim du, ngi ta dng

nhiu phn v c tng hp N, P, K ri xung ni nhim nhm kch thch s

pht trin ca vi sinh vt [4].

Cc vi sinh vt khc nhau s dng cc hyrocacbon khc nhau. Trong cc

hydrocacbon, hydrocacbon no v hydrocacbon thm b phn hu nhiu nht tt

nht.

Cc c tnh ca mn khoan c nh hng ti kh nng phn hu ca visinh vt. Mn khoan l mt cht thm m DDK - l mt dung dch kim tnh

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cao, cho nn mn khoan thng rt kim. Mn khoan l mt hn hp rn, cha

cc cht km hm vi sinh cho nn lng O2 trong mn khoan rt t v kh nng

sinh sng ca vi sinh vt trong khng thun li. iu ny gy nn nhng

kh khn nht nh trong vic x l chng. V vy hng s dng chnh vi sinh

vt phn lp t mn khoan v vic dng CHHBMSH kch hot qu trnhphn hu ca chnh tp on vi sinh vt trong mn khoan m bo kh nng

thnh cng.

2.3. Cc phng thc x l sinh hc

* nh gi mc nhim du: trong t nhin nhim du c gim i

bng nhiu cch nh s bay hi ca cc phn on nh hay s t oxy ho v

quang ho nhng c ch ch yu ca s phn hu du l nh vi sinh vt. iu

ny l c s cho cc nh vi sinh vt dng cc ch phm sinh h tr cc kthut lm sch ho l khc [12].

* Cc phng thc x l nhim du bng bin php phn hu sinh hc:

Khi nim phn hu sinh hc hay cn gi l cu cha sinh hc

(bioremediation) a ra m t qu trnh s dng cc vi sinh vt sng x l cc

h thng b nhim. Trong cc vi sinh vt th vi khun c s dng nhiu nht,

tuy nhin nm v cc thc vt bc cao cng gp phn x l nhim.

Bn cht ca qu trnh phc hi sinh hc l c gng thc y s phn hu

sinh hc trong t nhin. Khi trong mi trng b nhim du, cc vi sinh vt c

kh nng s dng du lm thc n lp tc pht trin. Tuy nhin s thnh cng

ca vic ng dng chng x l nhim du ph thuc vo kh nng ti u

ho cc iu kin khc nhau v sinh hc, ho hc, a cht gip cho vi sinh

vt sinh trng v pht trin mnh m nht c th, vi thi gian nhanh nht.

C hai phng thc x l nhim du trong t:

1. Bioaugmentation l phng thc x l m trong ngoi vic ti u ho

cc iu kin dinh dng, ngi ta a vo mt s vi sinh vt chn lc c kh

nng phn hu cao, b sung cng vi qun x sinh vt bn a trong mi trng

b nhim tng cng kh nng phn hu du.

2. Biostimulation l phng thc x l ly qun x sinh vt bn a lm

trung tm, cc c im sinh hc, sinh thi ca qun x vi sinh vt c nghin

cu k qua a cc cht dinh dng hu c, v c, cng cc ch phm sinh

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hc vi mt t l thch hp nhm kch hot tp on vi sinh vt c kh nng phn

hu du bn a lm sch mi trng.

Trn th gii c hai phng thc ny cng c em ra p dng nhng

theo cc ti liu th phng thc th nht dng nh t hiu qu hn khi x l

du trong mi trng m. Trong cc th nghim thc t, vic cho thm cc vivinh vt cho thy khng c g hiu qu hn so vi ch thm cc cht dinh dng.

Nguyn nhn c th l do khi a mt lng vi sinh vt mi vo mi trng,

chng phi mt thi gian thch nghi vi mi trng sinh thi mi, v thng

mt i kh nng phn hu mnh nh vn c do c cc tc nhn km hm no

trong mi trng nhim, nhiu khi chng chuyn sang s dng ngun c cht

mi khng phi l du. iu ny tht bt li, thm ch cn tr qu trnh phn

hu t nhin do chng ln t h vi sinh bn a, s dng ht ngun oxy vn lmt nhn t gii hn cc k nhy cm ca qu trnh phn hu. Kt qu l va

tn km m hiu qu li khng cao. Tuy nhin cc nh khoa hc cho rng

phng thc Bioaugmentation c tim nng trong vic x l cc hp cht du

c bit hoc l nhng vng m vi sinh vt bn a lm vic khng hiu qu

nh cc vng t si, t ct. Nga s dng ch phm Devoroie gm hn

hp cc vi khunRhodoccocus v Candida lm sch nhim du vng t ct.

Biostimulation c chng minh l mt cng c hu hn x l trit cc b bin b nhim du theo phng php hiu kh. Mt nhn t ch cht

to nn s thnh cng ca phng thc ny l duy tr mc dinh dng ti u.

Cc yu t km hm phi c kho st k lm c s cho quyt nh hm lng

dinh dng c a vo. Trong trng hp hm lng du cao th chng ta c

th b sung vi khun v mt s ch phm khc nh cht hot ha b mt tng

tc phn hu sinh hc Cc chng vi khun c b sung y phi l cc

chng c phn lp t chnh mi trng b nhim c nhn ging v bsung di dng ch phm.

Cc ch phm khc nhau khi a vo x l nhim cho cc kt qu khc

nhau ph thuc vo c tnh ca du, hm lng dinh dng t nhin sn c ca

vng b nhim, c im ca mi trng b nhim. Da trn cc th nghim

tht bi trong thc t ni chung cc ch phm thng mi c hiu qu hn cc

loi phn bn nng nghip trong vic thc y s phn hu du.

Hin nay nh s pht trin mnh m ca sinh hc phn t, s pht trin caphn loi hc, cc nh khoa hc c th gim st qu trnh phn hu sinh hc

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thng qua s bin ng s lng ca qun th vi sinh vt chim u th trong

mi trng b nhim, nh phng n phc hi sinh hc mi trng b

nhim du tr nn hp dn cc nh qun l nh gi r, an ton, tin cy cao.

3. VAI TR CA CHT HOT HO B MT SINH HC (BIO-SURFACTANT

HAY MICROBIAL SURFACE ACTIVE AGENT)3.1. Bn cht ca cht hot ho b mt sinh hc

Cht hot ho b mt sinh hc (CHHBMSH) c to ra t con ng ln

men vi sinh vt. N l mt hp cht lng tnh gm hai phn phn k nc

(hydrophobic moiety) v phn a nc (hydrophilic moiety), c kh nng lm

gim sc cng b mtgia cc phn t.

Phn a nc thng l cc nhm: axit amin, peptit, sacarit n, i,

polysacarit.

Phn k nc l cc axit bo, axit no, khng no [30].

Cc CHHBMSH c cu to rt khc nhau v cu trc ho hc ln kch

thc phn t t rt n gin nh cc axit bo n phc tp nh cc hp cht

polymer.

Cc CHHBMSH thng tit ra bn ngoi t bo (extracellular) nh cc

cht glucolipid, axit bo, photpholipid, polysacarit lipid, lipopetic-lipoprotein,

hay chnh bn thn b mt t bo vi sinh vt [30].

Ngoi c tnh lm gim sc cng b mt n cn c c tnh khng sinh

nh cht gramicidin S hay polymicin [30].

Cc CHHBMSH c to ra c trn cc c cht khng tan trong nc ln

tan trong nc [16,30]. N c to ra do phn ng thch nghi vi mi trng

khng thun li, c hi v c xu hng to ra nhiu trn cc c cht khng ho

tan trong nc [16].Cc CHHBMSH do cc gen trn nhim sc th ln cc gen plasmid tng

hp v iu khin. Cc gen ny d mt i chc nng qua mt thi gian di do b

t bin v chn lc nu gp mi trng khng thun li cho chng biu hin

[16]. iu ny gii thch v sao chng ta bt gp cc loi c kh nng s dng

du c nhng ni khng b nhim du v khi hot tnh to CHHBM rt

thp.

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Cc CHHBMSH to ra trong sut qu trnh pht trin ca vi sinh vt, sn

lng v hot tnh v sau c th b gim do mi trng ht ngun c cht dinh

dng, vi vinh vt s dng chng lm ngun thc n hay cc vi sinh vt cht i.

3.2. Cc loi CHHBMSH

CHHBMSH l mt nhm rt a dng, da vo cu trc ho hc c th chiachng thnh cc nhm sau:

3.2.1. Glucolipid: l mt nhm c nghin cu, ng dng nhiu nht v

c hot tnh mnh, chng gm 3 loi:

* Rhamnolipid: do mt hoc hai phn t rhamnoza lin kt vi mt hoc

hai phn t axit hydroxydecacnoic, gm c 4 loi: 2 phn t ng lin kt vi

2 phn t axit, 2 phn t rhamnose lin kt vi 1 phn t axit, 1 phn t ng

lin kit 2 phn t axit, v 1 phn t ng lin kt vi 1 phn t axit.

Vi sinh vt sn sut lPseudomonas aeruginosa

Cng thc cu to:

O

O CH

(CH2)6

CH3

CH2 C

O

CCH2CHO

O

OHH O

OH HO 3CH

(CH2)63CH

CH3

OHHO

OHO

O

CH3 (CH2)6

CH3

OHHO

O

O CH CH2 C

O

CCH2

3CH

(CH2)6

CHO

O

HO

Rhamnolipid

Rhamnolipid

* Trehaloselipid: do mt phn t ng i trehalose lin kt v tr C6 v

C6 vi cc phn t axit mycolic.

Axit mycolic l mt axit bo mch di c phn nhnh. Cc loi vi sinh vt

khc nhau to ra cc cht c mch hydrocacbon c chiu di v s lin kt i

cng khc nhau [30].

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Cc vi sinh vt nh nhiu thnh vin ca chiMycobacterium to ra cc este

trehalose trn b mt t bo [7]. Cc loi thuc cc chi Mycobacteria, Nocardia,

Brevibacteria to ra cc trehaloselipid khc nhau v cu trc v kch c. [9]

* Sophorolipid

Do 1 phn t ng sophorose lin kt vi mt axit bo mch di.

Phn t ng do hai phn t glucose lin kt vi nhau, nhm OH v tr

C6 v C6 b acetyl ho.

Chng do nhiu chng nm men thuc chi Torulopsis sn xut: T.

bombicola, T. petrophilum, T. apicola v loi Candida bogoriensis. Sophorolipid

c kh nng lm gim sc cng b mt nhng n khng phi l cht nh ho

hiu qu [9,24].

3.2.2. Cc axit bo: c to ra do cc vi sinh vt oxy ho ankan, gm c

cc axit bo mch thng v cc axit bo phc hp cha nhm OH v nhnh

ankyl. Cn bng du v nc (hydrophilic-lipophilic balance) ph thuc vo

chiu di ca mch cacbon. [36]

Chng do mt s vi khun v mt s nm men sn xut.

3.2.3. Phospholipid

Do mt s vi khun v nm men sn xut, n l thnh phn chnh ca mngvi sinh, khi vi sinh vt c nui trn cc c cht l ankan th hm lng

phospholipid tng mnh. Chng Acinetobater sp., Thiobacillus thioxydans,

Athrobacter AK-19, Pseudomonas,Aeruginosa 44T1, Aspergillus spp. c kh

nng sn xut phospholipid.

3.2.4. Cc lipid trung tnh

3.2.5. Cc lipopetid v cc lipoprotein

Cc cht va c c tnh khng sinh va c hot tnh b mt nhgramicidin S doBacillus brevis, polimicin doBrevibacterium polymixa tnghp.

[28]

Cc lipid cha ornithine doPeudomonas rubescen, Thiobacillus thioxydans,

cc lipid cha lysin doAgrobacterium tumefaciens IFO to ra.

Loi Seratia marcescens sn xut ra mt loi aminolipid c hot tnh b

mt.

ChngBacllus subtilis ATCC21332 xc nh c kh nng to ra mt loi

lipopeptid c hot tnh cao.

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3.2.6. Polymer

Hu ht cc CHHBMSH thuc nhm polymer l hn hp ca polysacarit

vi protein, chng l nhng cht c hot tnh b mt c khi lng rt ln v

cu trc phc tp. Cc cht tt nht l emulsan, liposan, manoprotein v mt s

phc hp ca polysacarit-protein khc.Emulsan do Acinetobacter calcoaceticus RAG-1 to ra, n phn ca n

cha mt trisacarit lin kt vi hai axit bo, n c kh nng nh ho cao cc

hydrocacbon trong nc thm ch vi nng rt thp [10].

Liposan to ra t Candida lipolytica, l mt cht nh ho ngoi bo, ho tan

trong nc, trong cu trc ca n cha 17% protein v 73% hydrocacbon, cc

hydrocacbon trong liposan gm c glucose, galactose, galactosamin, axit

galactorunic [12].

Manoprotein do Sacharomyces cerevisiae to ra cha 44% manose v 17%

protein.

Candida tropicalis sn xut ra mt loi phc hp manan-axit bo trn

mi trng cha ankan [25].

P. aeruginosa P-20 to ra mt peptidoglycolipid cha 52 axit amin, 11 axit

bo, 1 ng [16].Cyanobacterium phormidium J-1 to cc cht CHHBM l biofloculan v

emulcyan [16].

3.2.7. Cc CHHBMSH c bit

Mt s loi to ra cc ti ngoi bo c tc dng ct hydrocacbon thnh tng

phn nh, ng vai tr quan trng trong vic vn chuyn cc hydrocacbon vo

trong t bo. Cc ti do chng AcinetobacterHO1-N c ng knh 20-50 nm

cha protein, phospholipid v lipopolysaccharid, cha 5 phn phopholipid,khong 350 phn polysaccharid [26].

Hu ht cc vi khun phn hu hydrocacbon v gy c c cha cc

CHHBMSH l cc thnh trn b mt t bo, chng bao gm cc cu trc nh l

M protein.

3.3. Khi qut qu trnh to CHHBMSH ca vi sinh vt c th bit r c c ch r rng qu trnh to cht hot ng b mt

sinh hc ta phi theo di c sn phm ca tng giai on trao i cht ca vi

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sinh vt. Nhng trong phm vi ca bn kho lun ny ch xin c m t mt

cch s lc v qu trnh to ra cht hot ng b mt sinh hc ca vi sinh vt.

Nhn chung, mi mt loi vi sinh vt c kh nng to cht hot ng b mt

sinh hc li c mt c ch chuyn ho ring. Bi vy nu ni mt cch c th th

phm vi s l qu rng. Ta s xt c ch ca qu trnh to cht hot ng b mtsinh hc ca vi khun hiu kh.

Vi loi vi khun hiu kh, con ng chung l trnh t oxy ho cc

hydrocacbon thnh ru, sau n andehit, cc axit bo, ri n axetyl-CoA

hoc axit pyruvic. T y tip tc con ng chuyn ho trong chui h hp ca

vi sinh vt (chu trnh TCA), to ra cc axit sn phm c nhm chc a nc

(nhm OH hoc C=O). Cc sn phm ca chu trnh TCA sau kt hp vi cc

thnh phn ca du th to ln sn phm l cc axit amin hoc cc lipid chungtnh gm c mt u a nc (OH hoc C=O) v mt u a du (-CH2). Chnh

cc cu t ny l cc cht hot ng b mt sinh hc.

3.4. Cc vi sinh vt c kh nng to CHHBMSH

Trong t nhin c rt nhiu cc vi sinh vt c kh nng to CHHBMSH.

S to thnh CHHBMSH v chc nng ca cc cht ny thng c lin quan ti

s phn hu cc hydrocacbon. V vy, cc cht ny ch yu l do cc vi sinh

vt c kh nng s dng hydrocacbon sinh ra. Tuy nhin, mt s vi sinh vt

khc li s dng cc ngun c cht l cc hp cht hu c tan trong nc

(glucoz, glycerol, etanol) cng c kh nng sinh ra CHHBMSH. Mt s vi

sinh vt dng nh to ra cht ny nhm thch nghi vi cc iu kin mi

trng sng c bit ca chng, chng hn nh: trong cc b cha du, trong

t, trong i dng

Ngi ta phn lp c chng Pseudomonas aeruginosa trong nc

bm p vo ging khoan du th Venuezuela [19]. Chng ny c kh nng

thch nghi vi cc iu kin khc nghit trong b cha du v hn na, cc

CHHBMSH (rhamnolipit) do chng ny sinh ra li khng b cc iu kin c

bit trong ging khoan (pH, nhit , mn, Ca2+ v Mg2+) lm mt hot tnh.

ChngBacillus SP018 c kh nng to CHHBMSH ngay trong iu kin k kh

500C v chu c nng NaCl n 10%. Chng Bacillus AB-2 v Y12-B

c phn lp t ct nhim du c kh nng sinh trng trong mi trng c

cha hydrocacbon nhit 500C.

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T cc mu t nhim du phn lp c mt s chng vi sinh vt khc

nhRhodococcus,Bacillus pumilus [27], Arthrobactersp. MIS 38 [33]. T cc

vng nhim nhin liu phn lp c cc chng nh Ochrobacchum

anthropii, hay t cc vng bin b trn du phn lp c Pseudomonas

aeruginosa. Cc chng ny khng nhng c kh nng phn hu du m cn ckh nng to CHHBMSH cao.

Jeneman v cng s phn lp c chng Bacillus licheniformis JF-2

trong mt ging khoan du vng Carter, Oklahoma. Chng ny c kh nng

pht trin trong mi trng k kh v lm gim sc cng b mt ca mi trng

cn 30mN/m. Chng ny cng c th sinh trng trong mi trng c nng

NaCl ln ti 10%, nhit nui cy l 500C v pH dao ng trong khong t

4,6 n 9,0. Hn th na, chng khng b c ch bi s c mt ca du th. Vvy, chng ny c tim nng s dng trong phng php tng cng thu hi

du nh vi sinh vt.

Ngi ta c tnh, trong cc ging du M, khong 50-70% cc vi sinh

vt c th sinh trng c pH t 4-8, nhit di 75 0C, nng NaCl

khong 10% v c th sinh trng c c trong iu kin k kh hoc vi hiu

kh [19].

CHHBMSH do vi sinh vt sinh ra c th l cht ngoi bo hay chnh bnthn t bo, trong mt s trng hp cc cht ny c tnh khng sinh, do

chng c th phn hu mng t bo ca mt s vi sinh vt cnh tranh ngun

cacbon vi chng. Cooper pht hin chng Clostridium pasteurianum sinh mt

loi m chung tnh ngoi bo, cht ny c th lm gim sc cng b mt ca

mi trng t 72mN/m xung cn 55mN/m. Yakimov v cng s [38] phn

lp c chng Bacillus licheniformis BAS50, chng ny c th sinh trng k

kh trn mi trng c b sung ng glucoz v 0.1% NaCl v sinhCHHBMSH c tn l lichenysin. Kinsinger v cng s [21] pht hin mt

chng Bacillus subtilis c kh nng sinh CHHBMSH ngoi bo c bn cht l

surfactin, cht ny c kh nng khng vi nm.

Cc loi vi sinh vt khc nhau c th to ra cc loi CHHBMSH c bn

cht ho hc v trng lng phn t khc nhau. Ngi ta tch chit v m t

c bn cht ho hc ca mt s CHHBMSH do mt s chng vi sinh vt to

ra. Banat tng kt c mt s vi sinh vt c kh nng sinh CHHBMSH vbn cht ho hc ca tng cht do tng chng sinh ra trong bng sau:

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19/54

Tn vi sinh vt Tn CHHBMSH c to thnh

ArthrobacterRAG-1 Heteropolysaccarit

ArthrobacterMIS38 Lipopeptit

Arthrobacter Trechaloz, xaccaroz, fructoz

Bacilluslicheniformis JF-2 Lipopeptit

Bacillussubtilis Surfactin

Bacilluspumilus A1 Surfactin

Bacillus sp. AB-2 Rhamnolipit

Bacillus sp. C-14 Hirocacbon-lipit - protein

Candidaantarctica Mannosylethritol lipit

Candidabombicola Sophoroz lipitCandidatropicalis Mannan

Candidalipolytica Y-917 Sophoroz lipit

Clostridiumpasteuriannum Lipit chung tnh

Corynebacteriumhirocarbolastus Protein-lipit-cacbohirat

Corynebacteriuminsidiosum Photpholipit

Corynebacterium lepus Axit bo

Chng MM1 Glucoz, lipit, v axit hiroxidecanoicNocardiaerythropolis M chung tnh

Ochrobacchumanthropii Protein

Penicillumspiculisporum Axit spiculosporic

Pseudomonasaeruginosa Rhamnolipit

Pseudomonasfluorescens Lipopeptit

Phaffiarhodozyma Cacbohirat-lipit

3.5. Cc yu t nh hng ti kh nng sn xut CHHBMSH

a. Ngun cacbon

Cht HHBMSH c sn xut bi nhiu cc sinh vt khc nhau v chng

cng c nhng cu trc rt khc nhau, tuy nhin c mt s cc hin tng chung

lin quan ti vic tng hp chng:

Th nht l vic sn xut CHHBMSH c th do cc hydrocacbon hoc cc

c cht khng tan trong nc. Hin tng ny c nhc n bi nhiu tc gi

vi nhiu CHBMSH [30,16].

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Hin tng d thy khc l s km hm tng hp CHHBMSM bi gluco v

mt s cc cht trao i khc [16]. Trong trng hp ca Athrobacter

paraffineus I, CHHBMSH khng c to ra khi thay th hexan bng gluco

[22]. Mt chng thuc loi Pseudomonas aeruginosa sn xut ra mt cht

ging protein trn ngun hydrocacbon nhng khng to ra trn gluco, glycerol,axit palmitic [22]. Loi Torulopsis petrophilum khng to ra bt c mt

glucolipid no khi nui trn mi trng ng pha cha bt c ngun cacbon ho

tan trong nc no. i vi chng P. aeruginosa khc khi nui trn mi trng

cha glycerol nu thm gluco, acetat, succinat, citrat th sn lng rhamnolipid

gim mnh.

Nhiu loi vi sinh vt c bit l s tng hp nhiu loi CHHBM khc

nhau khi nui trn vi ngun cacbon [32].C nhiu v d v vic sn xut CHHBMSH trn cc ngun cacbon ho tan

trong nc nh Pseudomonas sp. sn xut CHHBMSH trn cc ngun

cacbon ho tan trong nc nh glycerol, gluco, manitol, ethanol [30].

Nhiu tc gi chng minh rng sn lng CHHBMSH to ra t khi nui

vi sinh vt trn cc ngun cacbon no trong mi trng, ch khi no ngun

cacbon ny ht, khi a vo ngun cacbon khng ho tan trong nc s thc y

to CHHBMSH [8]. Vic to glucolipid bi Torulospos bombiloca c kchthch khi thm du n vo trong mi trng nui cy cha 10% D gluco [16].

Davis cng cc cng s chng minh khi thm ethyl este ca mt loi axit bo

vo mi trng cha gluco th sn lng sopholipid chng C. bombicola

CBS6009 tng. Khi sn xut glucolipid chng T. apicola TMET73747, Stuwer

cng cc cng s thu c sn lng ln ti 90g/l trn mi trng cha gluco

v du hng dng [16].

b. Ngun nit

Ngun nit l mt yu t quan trng iu khin sinh tng hp CHHBMSH.

Ngun nit NH4 thch hp hn ngun NO3 cho vic to CHHBMSH chng

Athrobacter paraffineus ATCC 19558, ngun ure cng lm tng sn lng

CHHBMSH. Ngun NO3 to ra sn lng ln CHHBMSH P. aeruginosa v

Rhodoccocus sp. [16].

Sn lng CHHBM tng A. paraffineus khi thm cc axit amin nh: axit

glutaric, axit asparagin, hay glycin vo mi trng.

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Cu trc ca surfactin cng b nh hng bi nng L- aa to ra surfactin

Val-7 hay Leu-7 [16].

Vi chngB. lichenniformis BAS50 sn lng lichennysin A tng gp hai

n bn ln khi thm L-glutaric v L-asparagin vo mi trng.

Robert cng cc cng s chng minh rng NO3 l ngun nit tt nhtcho vic sn xut CHHBMSH chngPseudomonas 44TL vRhodococus ST-5

sinh trng trn du liu v paraffin.

Sn lng CHHBM cng tng bng vic gii hn ngun nit;

P. aeruginosa, Norcardia, C. tropicalis tng sn lng CHHBM cng

vi hot ng tng hp glutamic khi chuyn sang giai on sinh trng chm

li, lc m mi trng ht ngun nit.

Syldatle cng cc cng s chng minh rng vic gii hn ngun nit

khng ch lm tng sn lng CHHBMSH m cn lm thay i thnh phn ca

CHHBMSH.

Guerra-Santos cng cc cng s ch ra rng vic to rhamnolipit l cc i

khi t l C: N l 16:1 n 18:1, khng to ra khi t l ny l 11:1. [20]

c. Cc yu t khc

Mt s thay i trong mi trng nui cy cng lm tng sn lngCHHBMSH.

Kiu loi, sn lng v hot tnh CHHBMSH cng b nh hng khng ch

bi cc ngun cacbon khc nhau m cn bi nng , ngun nit, photpho, cc

ion Mg, Fe trong mi trng, iu kin nui cy, pH, nhit , tc lc v t l

pha long cc cht trong mi trng [21].

Trong mt s trng hp, khi thm cc ion a ho tr vo mi trng nh

hng dng tnh ti sn lng CHHBM.Mt s cc cht nh etabutol, penicillin, cloramphenicol, EDTA cng nh

hng ti vic to thnh CHHBM. l do cc yu t ny nh hng ti

ho tan ca cc ngun cacbon. [35]

Mt s trng hp qu trnh tng hp CHHBM li b nh hng bi nhit

, pH thng qua s tc ng ca cc yu t ny ti hot ng sinh trng ca

t bo.

pH ng vai tr quan trng trong vic to rhamnolipit Pseudomonas sp.,

CHHBMSH to ra cc i pH = 6 n 6,5 v gim mnh khi pH =7 [19].

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Kh nng to cellobioselipit Ustilago maydis [18] v to sophorolipit

Torulospos bombicola b nh hng mnh m bi yu t pH [13].

Trong trng hp chng Athrobacter paraffineus ATCC19558,

Rhodotorulaerythropolis, Pseudomonas sp. DSM2874 nhit li ng vai tr

quan trng. LoiBacillus chu nhit cao ch to CHHBM khi nhit trn 40oC.Tc lc cng nh hng ti sn lng CHHBM, chng Norcardia

erythropolis, sn lng CHHBM gim khi tng tc lc do n tc ng ti

hot ng phn chia t bo, cn nm men sn lng li tng khi tc lc v

mc k kh tng [29].

Nng mui cng nh hng nhiu ti qu trnh sn xut CHHBM do

nh hng ti hot ng ca t bo. a s cc trng hp khi nng mui

tng th kh nng to CHHBM gim. Tuy nhin trong mt s trng hp sn

lng CHHHBM khng b nh hng khi nng mui ln ti 10% mc d

nng mixen ti thiu gim nh [6].

3.6. Mt s ng dng ca CHHBMSH

* u im ca CHHBMSH so vi CHHBMHH

CHHBMSH to ra t con ng ln men sinh hc, cu to ca chng rt

khc so vi cc CHHBMHH, tuy nhin chng cng gm hai u k nc v anc v hot tnh ca chng cng khng km CHHBMHH. Nhng CHHBMSH

li c nhng u im m CHHBMHH khng th snh kp, quyt nh kh nng

ng dng ca n trong rt nhiu ngnh cng nghip, v ang c xu hng thay

th dn cc CHHBMHH. l cc c im:

- D phn hu sinh hc: CHHBMHH khi b sung vo trong m kh b

phn hu trong iu kin va, gy nhim mi trng du kh do ln cc cu t

khng mong mun lm gim cht lng du. CHHBMSH to ra t ngun visinh vt nn tinh khit cao, hot tnh ca chng li khng km cc

CHHBMHH. V d in hnh cho trng hp ny l nh hng ca CHHBMSH

v CHHBMHH ln s phn hu cc hp cht hydrocacbon thm mch vng

c nghin cu bi Marar v Rockne: khi thm mt s CHHBMHH vo c th

km hm s phn hu cc hp cht thm do c nh hng gy c, kch thch vi

sinh vt phn hu CHHBMHH hay c lp cc hydrocacbon trong mch micxen

CHHBM, cn CHHBMSH cho thy c nhng nh hng tt nh CHHBMHH

nhng chng c th b phn hu, khng c v nhiu CHHBMSH khng to cu

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trc micxen do rt thun li cho vic kt hp cc hp cht thm vi vi khun

[34].

- Cc cht HHBMSH c th sn xut t cc ngun vt liu r tin, sn c,

vi s lng ln. Ngun cacbon c th l cc hydrocacbon, lipit, cc ngun c

cht ph thi ca cc ngnh cng nghip khc nh r ng, du cn... do nmang li hiu qu kinh t cao m li x l c nhim do cc ph thi ny gy

ra vi mi trng [31].

- Vic sn xut CHHBMSH n gin hn nhiu CHHBMHH. Chng c

to ra bng con ng ln men nh vi sinh vt cho nn thit b n gin, tn

km m li khng s dng cc ho cht c hi nh sn xut CHHBMHH.

Do khng c, d dng b phn hu, gi thnh sn xut r nn chng c

ng dng trong x l mi trng m CHHBMHH khng th x l trit : x l

du trn, tng cng kh nng phn hu cc hp cht c hi cho mi trng,

chuyn chng thnh cc yu t tch cc cho mi trng nh sn xut phn bn

t mn khoan ph thi, cc hp cht thm.

Vi nhng c tnh khng c hi, d b phn hu, d tiu ho m

CHHBMSH c s dng trong cc ngnh cng nghip c bit khc nh m

phm, dc phm, thc phm m CHHBMHH kh c th b kp.

* Mt s ng dng ca CHHBMSH

CHHBMSH l mt nhm a cu trc, chng c nhng c tnh hn hn cc

CHHBMHH nh ni trn cho nn chng c ng dng trong rt nhiu

ngnh cng nghip khc nhau. Cng nghip du m l mt ngnh ng dng

rng ri cc cht hot ho b mt trong khai thc cng nh l x l mi trng,

CHHBMSH t ra c u th v tim nng trong ngnh cng nghip ny. Sau

y chng ti xin trnh by mt s cc ng dng quan trng ca n.

a. Nng cao hiu sut khai thc du (MEOR)

y l mt ng dng quan trng nht ca n trong ngnh cng nghip du

m.

C ch ca phng php MEOR bao gm:

1. To axit ho tan phn bao bc du, tng rng v tnh thm ca va,

to kh, do lm gim nht ca du v tng p sut va.

2. To cc axit hu c hay cc cht HHBM, biopolyme lm gim sc cngb mt gia du v nc dn n lm tng linh ng ca du.

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CHHBMSH c th c sn xut t ngoi (ex situ) ri b sung vo trong

m hay to ra ngay trong m (in situ). Trong c hai trng hp, CHHBMSH u

lm tng lng du khai thc t m ln ti 60 n 70% m bng phng php

bm p thng thng ch khai thc c khong 30% ngun du trong m. S

c mt ca CHHBM lm gim sc cng b mt gia vng du nc do kchthch dng du chy.

sn xut CHHBM t ngoi, CHHBM c tin hnh ln men, thu

CHHBM hay n gin l c c dch nui cy v sau l bm xung ging.

Qu trnh sn xut ex situ phi c tin hnh vi s lng ln, di iu kin

ti u, cung cp y khng kh cho qu trnh ln men. Qu trnh ny c th

s dng cc c cht r tin nh cc ph thi ca cc ngnh cng nghip nn va

h gi thnh sn phm va gim nhim cho mi trng [31].Mt p dng hp l v hp dn hn l sn xut CHHBMSH ngay trong m

(in situ) bng cch cung cp cht dinh dng cho qun th vi sinh vt ni ti

trong m hay nui ging t bn ngoi trn cng vi mi trng dinh dng ri

bm vo trong ging. Tuy nhin khi sn xut theo cch thc ny thng gp

nhng kh khn nht nh nh hng ti s sinh trng ca vi sinh vt thm ch

km hm sinh trng v sinh tng hp CHHBM nh: nhit m qu cao, p

sut ln, pH, mn v kim loi nng, kh khn ln nht l thiu oxy v viclun chuyn cht dinh dng trong m. Do , phng php MEOR ny

thnh cng th phi la chn c cc chng c kh nng chu ng c cc

iu kin kh khn trong m.

Phng php MEOR c nhiu nc p dng thnh cng.

Clark cng cc cng s xc nh c khong 27% m du M thch hp

cho vi sinh vt pht trin v ng dng phng php MEOR. Phng php

MEOR cng c thng bo thnh cng trong nhiu nghin cu Nga, Cngho Sc, Rumani, M, Hungary, Balan, H Lan, Nam T.

Nc ta hin ng dng phng php ny, nm 1998 Li Thu Hin

cng cc cng s th nghim phng php MEOR trn hin trng khu vc

m du Bch H- Vng Tu v thu c kt qu tt, s lng vi khun kh

sunphat gim i, nng sut khai thc du tng ln c ging tng ti 200%.

b. Phn hu cc hydrocacbon

Mt ng dng quan trng khc ca CHHBMSH l trong x l mi

trng nhim hydrocacbon.

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- Phn hu cc hydrocacbon trong t:

Cc nghin cu v qu trnh phn hu hydrocacbon trong t c

thng bo rng di. Kh nng phn hu ph thuc vo s c mt ca cc vi sinh

vt phn hu, thnh phn hydrocacbon, O2, nhit , pH, nc, cc cht dinh

dng v trng thi vt l ca cc hydrocacbon. Khi thm CHHBM sinh hc haytng hp vo trong mi trng s lm tng tnh lu bin v ho tan ca cc

hydrocacbon, iu ny rt cn cho qu trnh phn hu.

Cc th nghim khi b sung CHHBMSH phn hu cc hydrocacbon

em li nhiu thnh cng:

Lindley v Heydeman nui nm Cladosporium resinae trn hn hp ankan,

chng ny c kh nng sn xut ra mt loi CHHBM gm cc axit bo,

phospholipid, nhng ch yu l axit dodecacnoic v phosphatidylcloline. Khi b

sung phosphatidylcloline vo mi trng nui cy cha hn hp cc ankan ny

lm tng tc phn hu ankan ln 30%.

Foght cng cc cng s chng minh cht emulsan kch thch qu trnh

phn hu ca dch cha n chng vi khun nhng li km hm khi mi trng

cha hn hp cc chng vi khun.

Oberbremer v Muller-Harting s dng qun th vi sinh t nh gi

kh nng phn hu du th, trong naphthalen b phn hu u tin, cc thnh

phn khc ch b phn hu khi cc CHHBM c to ra lm gim sc cng b

mt gia du v nc.

Khi thm CHHBM nh mt s sophorolipit vo mi trng cha hn hp

cc hydrocacbon th s lm tng phn hu c s loi ln s lng cc

hydrocacbon.

Mt vi chng vi khun k kh cng sn xut CHHBMSH nhng kh nnglm gim sc cng b mt khng bng cc vi khun hiu kh.

Bergetal s dng CHHBMSH do chngP. aeruginosa UG2 lm tng

ho tan ca hexachlorobiphenyl ln 31% gp 3 ln khi s dng CHHBM ho

hc l 9,3%. Khi c hai cht ny cng s dng th hiu qu ho tan tng ln

41,5%.

ChngPseudomonas cepacia AC1100 to ra mt loi cht nh ho c kh

nng to th huyn ph vi 2,4,5-T v cng nh ho mt s cc clorophenol, do

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CHHBM ny c s dng tng cng kh nng phn hu ca cc cht

hu c cha clo [30].

- Phn hu cc hydrocacbon trong mi trng nc:

Khi du b trn mi trng nc, cc thnh phn HRCB nh bay hi cn

cc HRCB khng phn cc th khng ho tan trong nc. Do c ho tan thp(


27/54

CHNG 2. VT LIU V PHNG PHP NGHIN CU

1. VT LIU

1.1. Nguyn liu

Mn khoan ly t m Bch H Vng Tu.

Cc chng vi khun nhn t phng vi sinh vt du m.

1.2. Ho cht v mi trng nui cy

1.2.1. Mi trng hiu kh tng s 1%(g/l)

Thch

KH2PO4

NaCl

KCl

MgCl2

NH4 NO3

Glucoza

Pepton

Cao tht

Cao men

Nc my

pH

20

1

10

0,25

1,2

2

1

5

3

0,2

1 lt

7-7,2

Vit Nam

Vit Nam

Vit Nam

Vit Nam

Vit Nam

Vit Nam

Trung Quc

Nga

c

c

1.2.2. Mi trng khong Gost cho vi khun (g/l):

Na2HPO4

KH2 PO4

KNO3

MgSO4

Nc my

0,7

0,3

3

0,4

1 lt

pH v nng NaCl chnh theo tng th nghim.

Cc loi du: DO, saralin, du th, parafin, du oliu.

Cc mi trng c kh trng ring vi cc loi du 1 atm trong thi

gian 30 pht.

1.3. Thit b v my mc

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Knh hin vi quang hc Laborval 4 (c)

Cn phn tch Sartorius (c)

My bay hi chn khng SpeedVac (c)

My li tm Sorwal (c)

My lc Beckman (c)

T sy (Vit Nam)

Bc cy (Vit Nam)

Cc vt dng khc ca phng vi sinh vt du m.

2. PHNG PHP NGHIN CU

2.1. Gi ging v nhn ging

* Gi ging: cc chng sau khi la chn c gi ging trn mi trngkhong Gost b sung du DO, gi trong glycerin vi t l 1:1, v trn mi

trng hiu kh thch nghing.

Nhn ging: chng c nhn ging trn mi trng hiu kh thch

nghing, ri t c cy sang mi trng Gost c b xung 5% du DO

tin hnh cc th nghim tip theo.

2.2. Tuyn chn cc chng c kh nng s dng du mnh

Cc chng sau khi phn lp t mn khoan du kh c nhn ging v v

cy chuyn ra cc ng thch nghing.

Tin hnh nui lc tng chng trn mi trng Gost 1% NaCl cha 5% du

DO, pH =7,5; tc lc v =200vng/pht vi t l 1% ging, theo di trong mt

tun. Quan st s thay i c, trng thi ca du trong mi trng, kt lun

chng c kh nng s dng du hay khng. Nu mi trng c c cao, du

b nh ho th chng t chng c kh nng s dng du mnh.

2.3. Quan st hnh thi

2.3.1. Quan st hnh thi khun lc: cy ria cc chng trn mi trng

hiu kh, iu kin 30o C sau 24h em quan st.

2.3.2. Quan st hnh thi t bo: sau24h nui cy, tin hnh nhum Gram

quan st hnh thi t bo vi khun bng knh hin vi quang hc.

* Xc nh Gram: nhum Gram v th li bng phng php KOH lm

tng chnh xc: ly mt t sinh khi t bo ln phin knh, nh mt git dungdch KOH 3% ln sinh khi t bo, dng que cy nh tan t bo. Nu dch to

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nht th kt lun vi khun thuc nhm Gram m, nu khng to nht chng t vi

khun l Gram dng

* Xc nh kh nng chuyn ng: lm tiu bn git p v soi knh hin vi.

2.4. Xc nh cc c im sinh ho bng cc php th ho sinh : nhm

mc ch xc nh kh nng s dng mt s cc c cht ca chng nghin cu.

2.5. Xc nh s lng t bo trn mi trng thch (phng php

Koch)

Pha long dch theo dy thp phn: Chun b mt lot cc l penicilin cha

4,5 ml mui sinh l v trng, ht 0,5 ml dch cn pha long vo l penicilin th

nht v lc u, t l th nht chuyn 0,5 ml sang l penicilin th hai, c thc

hin lin tip nh vy cho n pha long thch hp.

Sau khi pha long ht 0,1ml dch pha long thch hp vo a cha

mi trng hiu kh, dng que trang gt u. Trong qu trnh pha long tin

hnh vi hai pha long chn hoc l lin tip thch hp, mi pha long

ht vo hai a peptri gim sai s, nui nhit 300C sau 1-2 ngy em xc

nh s lng t bo.

Cng thc tnh s lng t bo:

X = n * 10(x+1)

Trong : X l s lng t bo trong 1ml dch cn xc nh.

n l s lng t bo trung bnh cc a

x l sth t ca l pha long

2.6. nh gi kh nng sinh CHHBM theo phng php Pruthi

Kh nng sinh CHHBM c nh gi thng qua ch s nh ho E24

Ch s nh ho E24 c trng cho kh nng nh ho ca cc sn phm trao

i cht do vi sinh vt sinh ra trong dung mi xylen sau 24h trong 4oC.

Cch tin hnh: Ly 1ml dch nui cy ly tm loi b t bo thm 1 ml

xylen vo ng nghim em voltex trong 1 pht vi vn tc 2000 vng/pht.

Mu c gi 4oC trong 24h ri em o.

Cng thc xc nh ch s E24:

Chiu cao ct nh hoE24 = x100%

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Chiu cao tng s

E24 cng cao th kh nng nh ho ca sn phm cng ln.

2.7. Ti u ho mt s cc yu t nh hng n qu trnh to

CHHBM theo phng php Gause- ZendenPhng php Gause-Zenden l phng php ti u n gin, c tin

hnh bng cch ch thay i mt yu t tc ng, c nh cc yu t tc ng

cn li. Qu trnh A chu nh hng ca n yu t, ch s cn ti u l y.

C nh (n- 1) yu t v cho mt yu t thay i trong khong xc nh

cng ngh ca n, ng vi mi mt th nghim ta xc nh c mt s liu y.

Trong dy s liu thu c ta xc nh c mt gi tr y ti u. Ta thu clot th nghim th nht.

Lot th nghim th 2 ta cng c nh (n-1) yu t tc ng mt gi tr

no , cho yu t th n thay i trong khong xc nh cng ngh. Trong dy

th nghin thu c ta c mt gi tr ti u ca yu t th 2.

Thc hin n lot th nghim nh vy ta xc nh oc cc gi tr ti u ca

cc yu t tc ng m ti y ti u.

Phng php ny ch tim cn ti vng ti u.

Trong phm vi kho lun ny, ch s cn ti u l sn lng CHHBM, cc

yu t tc ng l nhit , pH, nng mui v ngun cacbon.

2.8. Phn tch sn phm bng ph hng ngoi

Cu trc phn t ca cht hot ho b mt c nghin cu bng phng

php phn tch ph hng ngoi nh s gip ca phng ph hng ngoi Vin

Cng ngh ho hc d on cu trc ho hc ca sn phm.

2.9. Xc nh trng lng kh ca CHHBM

Phng php tch chit cht hot ho b mt c tin hnh theo phng

php Pruthi c ci tin. Dch nui cy vi khun sau 120 gi nui lc c dng

tch chit cht hot ho b mt sinh hc.

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Dch nui cy

Li tm 8.000 v/ph 10

Dch huyn ph CnB sung axeton t l 1:4 gi 4oC trong 24h

Li tm

Cht kt ta Dch nui cy v axeton

Lm kh chn khng

Cht hot ho b mt

Trng lng ca CHHBMSH c o bng cn phn tch.

2.10. Xc nh bn hot tnh ca CHHBM

Dch nui cy c li tm 20.000 vng/pht trong thi gian 10 pht loi

b ton b t bo, thu phn dch cha CHHBM.

Phn dch thu c t cc nhit 30oC, 55oC trong thi gian 3 ngy,

10 ngy, 15 ngy v nhit 100o

C trong 30 pht, 60 pht; ri em o hottnh nh gi bn ca CHHBMSH.

2.11. nh gi nh hng ca CHHBMSH ln mn khoan

* Lm giu vi khun phn hu saralin trong mn khoan:

Cn 10 gam mn khoan cho vo bnh 250ml cha mi trng Gost c b

sung 5% du saralin, tin hnh lc trong 3 ngy. Sau cy chuyn 2ml dch

nui lc sang bnh khong Gost th hai cng b sung 5% du saralin, lc trong 5ngy. Hn hp vi khun phn hu du c dng lm ging.

* B sung CHHBMSH lm tng kh nng phn hu du:

Nui cy chng M150 thu c lng CHHBM cc i.

Thu CHHBM bng cch li tm loi b t bo, thu dch nui cy.

Chun b 4 bnh khong Gost cha 5% saralin, b sung vo cc bnh 0,5ml

dch hn hp vi khun phn hu du.

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33/54

CHNG 3. KT QU V THO LUN

1. MT S CHNG VI KHUN SINH CHHBMSH

1.1. Tuyn chn chng c kh nng s dng du mnh

T b su tp gm 16 chng vi khun phn lp c trong mn khoan dukh m Bch H Vng Tu, chng ti tin hnh kho st kh nng s dng du

ca chng. Th nghim c tin hnh trn mi trng khong Gost c b sung

5% du DO, lc nhit 30o C trong 7 ngy. Kh nng s dng du c nh

gi nh tnh thng qua c ca mi trng v s thay i trng thi vt l

ca cc git du. T , chng ti tm c 3 chng c kh nng s dng du

mnh l M150, MT3 v MX. Cc chng ny la chn cho cc nghin cu tip

theo.1.2. c im hnh thi ca mt s chng phn hu du

Cc chng vi khun ria trn a cha mi trng hiu kh tng s 1% NaCl,

trong t m 30o C. Sau 24 gi nui cy em ra quan st hnh thi khun lc,

ng thi cng tin hnh nhum Gram xc nh hnh thi t bo, lm tiu bn

git p quan st kh nng chuyn ng. Kt qu c th hin Bng 1.

Bng 1. c dim hnh thi ca mt s chng vi khun phn lp

c kh nng phn hu du

K hiu

chngc im khun lc c im t bo

MT3 Trn dt, mp khng u, mu

vng nu, hi dt, khun lc to

2,5 -3 cm.

Que ngn, Gram m, khng c

kh nng chuyn ng

MX Trn u, mu xanh l cy,

bng, li, khun lc nh 1cm.

Que ngn, Gram dng, khng

c kh nng chuyn ng.

M150 Trn u, bng li, mu vng,

nh 1 cm.

Que ngn, hi cong, Gram

dng, khng c kh nng

chuyn ng.

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Hnh1. Hnh thi khun lc chng M150 trn mi trnghiu kh tng s 1% NaCl

1.3. Kh nng sinh CHHBMSH ca cc chng vi khun phn hu du

la chn tip chng ti tin hnh nh gi s b kh nng to CHHBM

ca cc chng ny. Kh nng to CHHBMSH c xc nh bng phng php

Pruthi - xc nh ch s nh ho E24.

Cc chng c nui lc trn mi trng khong Gost cha 5% (v/v) du

DO. Theo di lin tc v o ch s nh ho E24 sau 5 ngy nui lc, kt qu cho

thy chng M150 c ch s nh ho cao nht l 69,8%, chng MT3 l 59%,

chng MX l 63%, c bit chng M150 c tc to CHHBM nhanh nht ch

sau 3 ngy nui lc.

Bng 2. Kh nng sinh CHHBM ca cc chng vi khun

Chng E24 (%)

M150 69,8MT3 59,0

MX 63,0

Chng M150 c la chn nghin cu su hn.

Theo kt qu phn tch trnh t 16S rARN xc nh chng M150 thuc

loiBrevibacterium celere. Loi ny mi c pht hin vo nm 2004 [17], do

, cc nghin cu v chng cn rt hn ch. Do vy phc v cho cc bctip theo chng ti tin hnh nghin cu mt s cc c im sinh l sinh ho

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ca chng ny.

2. MT S CC C IM SINH L SINH HO CA CHNG M150

2.1. Mt s c im sinh ha

phc v cho cc mc ch xy dng mi trng ti u cho s sinh

trng cng nh s to CHHBM ca chng M150 trong cc giai on sau ny

chng ti tin hnh kho st s b kh nng s dng mt s ngun c cht bng

cc php th ho sinh.

Kh nng s dng mt s c cht ca chng M150 c th hin Bng 3.

Bng 3. Kh nng s dng mt s cc ngun c cht ca chng M150

STT Ngun c cht Kh nng

1 NO3 + +2 Tryptophan -

3 Gluco (k kh) -

4 Arginin + +

5 Ure + +

6 Esculin -

7 Gelatin -

8 Gluco + + +9 Gluconat +

10 Caprat -

11 Adipat +

12 Malat +

13 Citrat -

14 Phenylacetat -

Trong : +++ l c kh nng s dng mnh; + l s dng yu++ l s dng trung bnh; - l khng c kh nng s dng

Vy chng M150 c kh nng kh nitrat thnh nitrit, s dng arginin, ure,

gluco iu kin hiu kh; s dng yu gluconat, adipat, malat; chng khng c

kh nng, gelatin, trytophan, escunin, caprat, ciprat v phenylacetat, gluco trong

iu kin k kh

2.2. ng thi sinh tng hp CHHBM ca chng M150

CHHBMSH l mt sn phm ca qu trnh trao i cht chng c to ra

trong sut qu trnh sinh trng ca vi khun. Theo cc tc gi th lc u cht

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hot ho b mt tng dn, n mt thi im no n gim dn do mt s cc

vi sinh vt s dng chng lm ngun thc n, hay vi sinh vt b cht i (i vi

CHHBMSH l chnh cc t bo vi sinh vt). c th thu c lng CHHBM

cao nht chng ti tin hnh xc nh ng thi sinh tng hp CHHBMSH ca

chng nghin cu.Chng c nui lc trn mi trng khong Gost, pH = 8, 30 oC, b

sung 5% du DO, theo di v o lin tc ch s nh ho E24. ng thi sinh tng

hp CHHBM ca chng c th hin Hnh 2.

0

0.2

0.4

0.6

0.8

0 24 48 72 80 96 104 120 168

Thi gian (h)

Chsnh

ho

Hnh 2. ng thi sinh tng hp CHHBM ca chng M150Kt qu t th cho thy chng bt u to CHHBM t 48 gi v tng

dn ln, t 96 gi n 120 gi cao nht t ch s nh ho72.7% v khng tng

na, n 168 gi gim dn.

Nh vy sau 4 ngy chng cho lng CHHBM cao nht. Khong thi gian

sinh CHHBM cao nht l tng i ngn so vi cc vi khun Gram dng.

3. TI U HO IU KIN TO CHHBM

S to thnh CHHBM ca vi khun chu nh hng ca iu kin mi trngv dinh dng. Trong phm vi kho lun ny, chng ti kho st mt s cc yu t

quan trng nh pH, nhit , mn v ngun cacbon. Cc yu t ny tc ng ti

qu trnh pht trin v to CHHBMSH. Do chng ti tin hnh o s lng t

bo v ch s nh ho E24 qua cc thi im la chn c nhng thng s ph

hp cho s pht trin v to CHHBM ca chng M150.

3.1. Kho st s thay i ca pH mi trng

pH l yu t quan trng v c nh hng rt ln ti s sinh trng v pht

trin ca cc loi nn n c chn nghin cu u tin.

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Theo cc tc gi thng bo th vi khun thng c kh nng pht trin

tt nht pH trung tnh, thm vo chng ny c phn lp t mn khoan l

mt ngun cht c tnh kim nn chng ti la chn di pH kh rng t 6 n 9,

ngoi mc ch xc nh pH ph hp, chng ti cn mun nh gi phm vi

chu pH ca chng to iu kin thun li cho cc bc nghin cu v sau.Chng ti tin hnh chun b cc bnh cha mi trng vi cc pH 6; 7;

7,5; 8,5; 9 c b sung 5% (v/v) du DO lc vi vn tc 200 vng/pht iu

kin nhit phng. Kt qu c th hin Bng 4 v Hnh 3.

Bng 4. nh hng ca pH ln kh nng sinh trng v to CHHBM

Thi gian(gi)

Log s lng t bo (CFU/ml) Ch s nh ho E24(%)

6 7 7,5 8,5 9 6 7 7,5 8,5 9

0 4,8 4,8 4,8 4,8 4,8 0 0 0 0 0

48 6,1 6,7 7,2 6,1 5,3 0 0 4,4 0 0

72 6,6 - 8,6 6,2 5,4 0 22,2 26,6 9,0 2,2

80 7,0 7,9 9,1 7,5 6,9 0 54,4 59,0 13,2 4,4

96 7,6 8,2 9,7 8,3 7,2 4,4 63,5 59,0 54,4 31,1

104 7,7 - 9,0 - 7,7 9,0 68,1 72,7 68,1 59,0

168 - - - - - - 50,0 59,0 54,4 -

0

1

2

3

4

5

6

7

8

9

10

0 48 72 80 96 104 168

Thi gian (gi)

Logsl-ngtbo(CFU

0

10

20

30

40

50

60

70

80

Chsnhho

E2

pH 6 pH 7 pH 7.5 pH 8.5 pH 9

E24 6 E24 7 E24 7.5 E24 8.5 E24 9

Hnh 3. nh hng pH ln s sinh trng v to CHHBM ca M150

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Kt qu cho t Bng 4 v Hnh 3 cho thy pH =7,5 th cho ch s nh ho

cao nht E24 =72,7% ( 104 gi) ng thi vi s lng t bo cao nht ln ti

hn 10 9 CFU/ml. pH =6 th cho ch s nh ho cao thp nht E24 = 4,4%, pH =

7 cng cho ch s nh ho kh cao E24 = 68,1% vo 104 gi.

Nh vy pH = 7,5 l ph hp nht cho s pht trin v to CHHBM cachng vi khun. pH = 7,5 c s dng tip cho cc th nghim v sau.

3.2. Kho st s thay i nng mui NaCl mi trng

Do chng phn lp t mn khoan du kh m Bch H ngoi khi Vng

Tu nn vic kim tra mn c nh hng nh th no ti s pht trin ca vi

khun l rt quan trng cho vic ng dng v sau.

Chng ti chn cc th nghim vi di nng NaCl l 0%; 0,5%; 1%; 2%;

3% vi ngun cacbon l DO, pH =7,5, iu kin nhit phng. nh hng

ca vic thay i nng mui c biu hin Hnh 4 v Bng 5.

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Bng 5. nh hng ca nng mui NaCl n

sinh trng v to CHHBMSH

Thi

gian

(gi)

Log ca s lng t bo (CFU/ml) Ch s nh ho E24 (%)

C-0 C-0,5 C-1 C -2 C-3 E-0,5 E-0,5 E-1 E-2 E-3

0 4,6 4,6 4,6 4,6 4,6 0 0 0 0 0

24 5,4 6,3 5,8 5,2 4,8 0 0 0 0 0

48 6,3 6,8 7,0 5,4 5,0 0 0 0 0 072 7,0 7,9 7,3 6,0 5,4 0 26,6 0 0 0

80 7,3 8,6 7,8 7,2 6,1 0 36,5 4,4 0 0

96 8,2 8,9 8,4 7,8 6,7 4,4 59,0 13,2 0 0

Cc s liu cho thy nng mui C=0,5% th s lng t bo l cao

nht v cng chnh nng NaCl cho ch s nh ho cao nht E24= 59%. Cc

nng mui 0%, 3% cho ch s nh ho v lng t bo thp nht l 4,4% v

9%, nng mui 1% v 2% cng thp hn nhiu so vi nng mui 0,5%.

Vy nng mui 0,5% l thch hp nht cho s pht trin cng nh l s

to CHHBM ca M150. Nng mui 0,5% dng cho cc th nghim tip theo.

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2

3

4

5

6

7

8

9

0 24 48 72 80 96

Thi gian (gi)

Logsl-ngtbo

(CFU

0

10

20

30

40

50

60

70

Chsnhho

E2

C-0 C-0.5 C-1 C -2 C-3

E-0.5 E-0.5 E-1 E-2 E-3

Hnh 4. nh hng ca nng NaCl ln s sinh trng v

to CHHBM ca chng M150

3.3. Kho st s thay i nhit mi trng

Th nghim c tin hnh nng mui 0,5%; pH = 7,5; ngun cacbon

l du DO vi cc nhit l 22 oC, 30oC, 37oC; kt qu c th hin Bng 6

v Hnh 5.

Bng 6. nh hng ca nhit n s sinh trng vo to CHHBM

Thi gian(gi)

Log ca s lng t bo (CFU/ml) Ch s nh ho E24 (%)

T-22 T-30 T-37 E-22 E-30 E-37

0 4,3 4,3 4,3 0 0 0

48 6,3 7,0 5,8 0 0 0

72 7,1 7,6 6,3 0 0 0

80 7,7 8,4 6,8 0 31,1 0

96 7,5 8,4 7,5 4,4 59,0 2,2

104 7,6 8,6 7,9 13,2 72,7 4,4

168 7,7 8,3 6,6 35,5 63,4 13,2

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2

3

4

5

67

8

9

0 48 72 80 96 104 168

Thi gian (gi)

Logsl-ngtbo

(CFU/ml)

0

10

20

30

40

50

60

70

80

ChsnhhoE

24(%)

T-22 T-30 T-37

E-22 E-30 E-37

Hnh 5. nh hng ca nhit ti s pht trin v to

CHHBM ca chng M150

T s liu thu c Bng 6 v Hnh 5 cho thy, nhit thch hp nht

cho vic tng hp CHHBM l 30o C. Nhit cao hay thp hn cng nh hng

ti s sinh trng v to CHHBM ca chng th hin r rng nhit 22oC v

37o

C vi khun to sinh khi t v ch s nh ho thp.Vy nhit 30o C l ph hp nht cho s sinh trng v to CHHBM

trong s cc nhit nghin cu. Nhit ny c nhiu nh khoa hc

thng bo l ph hp cho s pht trin ca a s cc loi vi khun.

Nhit 30o C c s dng cho cc th nghim tip theo.

3.4. Kho st s thay i ngun cacbon

thu c CHHBM cao nht, ngun cacbon cng l mt yu t quantrng. Chng ti la chn cc ngun cacbon l DO, paraffin, du oliu l cc

ngun cacbon c thng bo l rt ph hp cho s to CHHBMSH v du

saralin l ngun cacbon trong mn khoan cn x l.

Tin hnh th nghim vi cc ngun cacbon DO, du saralin, paraffin, du

oliu iu kin nng mui 0,5%; pH=7,5; iu kin nhit phng. Kt qu

c th hin Bng 7 v Hnh 6.

Bng 7. nh hng ca ngun cacbon ti s

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pht trin v to CHHBM

Thi

gian

(gi)

Log s lng t bo (CFU/ml) Ch s nh ho E24(%)

DO Saralin Parafin Oliu DO Saralin Paraffin Oliu

0 4,9 4,9 4,9 4,9 0 0 0 0

48 6,8 7,1 5,9 6,3 0 0 0 0

72 8,2 8,7 7,3 7,1 17,7 9 0 0

96 9,6 9,5 8,5 7,1 54,4 41 13,2 0

168 8,3 4,2 8,4 7,2 50,0 35,5 31,2 0

3

4

5

6

7

8

9

10

0 48 72 96 168Thi gian (gi)

Logsl-ng

tbo(CFU

0

10

20

30

40

50

60

Chsnhho

E2

D DO D Saralin D Parafin D Oliu

E DO E Saralin E Paraffin E Oliu

Hnh 6. nh hng ca ngun cacbon ln kh nng sinh tng hp CHHBM

ca chng M150

Cc s liu cho thy du DO l ngun cacbon thch hp nht trong s cc

ngun cacbon kho st cho s to CHHBM ca chng M150 ng thi cho s

pht trin ca chng ch s nh ho t E24= 54,4 %.

Nh vy iu kin pH =7,5; nng mui 0,5%; nhit 30oC v du

DO l thch hp cho chng sinh trng v to CHHBMSH.

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S to thnh CHHBMSH cng song song vi qu trnh pht trin ca

chng khi E24 tng th s lng t bo cng tng theo. Kt qu thu c cng

hp l v CHHBM to ra nhm mc ch gip vi sinh vt s dng d dng cc

ngun cacbon khng tan trong nc nh cc ngun cacbon kho st.

4. MT S C IM CA CHHBMSH TO RA T CHNG M150

4.1. Cu trc CHHBMSH

Cu trc phn t ca cht hot ho b mt do chng M150 c nghin

cu bng phng php phn tch ph hng ngoi. Kt qu thu c biu din

trn Hnh 7 cho thy trong cu trc phn t ca cht hot ho b mt c cha

cc nhm chc -OH, CH2, CH.

Nhm OH vi cc v tr tng ng vi tn s giao ng v = 1653/cm,

v = 1079,09/cm, v = 3377,49/cm.

Nhm CH tng ng vi cc v tr c tn s giao ng l v = 1379,29/cm

Ti cc v tr c tn s giao ng l v = 1735,42/cm l gc du.

Cc nhm chc ny u c mt trong thnh phn ca cht hot ho b mt

sinh hc c c quan bo v mi trng ca M cng b. Trong cc nhm

chc ni trn th nhm -OH v ng vai tr l tc nhn a nc. Nhm CH, gc

du ng vai tr l tc nhn k nc.

Tuy nhin c th kt lun chnh xc CHHBM do chng to ra c thuc

nhm trehalolipit nh nhiu loi thuc chi Brevibacterium hay khng th cn

phi phn tch tip khi ph.

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Hnh 7. Ph hng ngoi ca CHHBMSH do chng M150 to ra

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4.2. Hm lng CHHBMSH

Sau khi xc nh c iu kin mi trng thch hp v thi im sinh

CHHBMSH cao nht, chng ti tin hnh thu CHHBMSH nh gi hm

lng cao nht ca n trong 1ml dch nui cy.

Sau khi lc120h iu kin pH =7,5; nng mui 0,5%; ngun cacbon l

du DO; nhit 30oC, tin hnh thu CHHBMSH bng cch kt ta vi

acetone, sy kh chn khng ri em cn bng cn phn tch c trng lng

kh ca CHHBMSH l 11,6 g/l.

4.3. bn ca CHHBM

Do iu kin thc t x l nhim nhit rt cao, c th ng dng

c, vic xc nh kh nng chu nhit ca CHHBM l rt cn thit.

bn ca CHHBM c nh gi thng qua nhit v thi gian lu gi

n.

Khi tin hnh gi nhit 100oC ch s nh ho cn 92,8% sau 30 pht

un v 78% sau 1h.

bn cn c nh gi 30oC v 55oC trong thi gian 3 ngy, 10 ngy,

15 ngy. Kt qu th hin Bng 8.

Bng 8. bn ca CHHBM do chng M150 sinh ra (%)

Thi gian

Nhit 3 ngy 10 ngy 15 ngy

30oC 100 100 92,8

55oC 100 89 80

Nh vy CHHBMSH tng i bn vi nhit: 30o C hot tnh khng my

thay i sau 15 ngy, cn 55o C hot tnh bin ng hn, 100oC hot tnh cnng k sau 30 pht.

5. NH HNG CA CHHBMSH N KH NNG PHN HU DU CA

QUN TH VI SINH VT TRONG MN KHOAN

Sau khi c nhng kt qu v c tnh ca CHHBMSH do chng M150

to ra. Chng ti tin hnh thu CHHBM, b sung vo trong mi trng cha

du saralin cng vi cc vi sinh vt s dng du trong mn khoan ng gi

kh nng kch hot cc vi sinh vt ny phn hu du saralin v lng CHHBM

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b sung c ngha nh hng. Kt qu thu c s nh hng cho chng ti

trong qu trnh x l vi quy m ln hn.

nh hng ca CHHBM c nh gi thng qua tc ng lm thay i

s lng vi sinh vt trong mn khoan. Khi s lng vi sinh vt tng ln c ngha

l cc chng ny s dng du lm ngun dinh dng v lng du gim. yl cch thc x l nhim du theo phng php biostimulation ly qun

x sinh vt bn a lm trung tm ca qu trnh x l nhim.

S bin ng s lng vi sinh vt c th hin Bng 9

Bng 9. nh hng ca CHHBMSH ln s lng vi sinh vt mn khoan

CHHBM b sung

(ml)

S lng vi sinh vt (CFU/ml)

Trc th nghim Sau th nghim

0 4,27.103 6,2.102

1 1,7.106

2 7,8.108

3 1,1.109

Kt qu cho thy khi b sung CHHBM do chng M150 vo trong mitrng cha du lm tng s lng vi sinh vt trong mn khoan nhim du. S

lng vi sinh vt tng ln rt cao ti 1 triu ln so vi ban u ch sau 4 ngy.

Trong khi th nghim khng b sung CHHBM th s lng vi sinh vt gim

i ti gn 10 ln. Khi b sung t 2 n 3ml CHHBMSH th c nh hng lm

tng r rt. 2ml s lng t bo cng tng ln rt ln bng 70,9% so vi khi

b sung 3 ml. Lng CHHBM b sung 3ml lm tng s lng vi sinh vt r

nht. S lng vi sinh vt tng ln ti 10

9

CFU/ml.Vy c th kt lun CHHBM do chng M150 sinh ra c tc ng kch thch

cc vi sinh vt trong mn khoan s dng du saralin. Lng cht b sung l 3ml

lm tng r nhtv c ngha ng dng.

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KT LUN

1. T 16 chng vi khun phn lp t mn khoan du kh Vng Tu la

chn c 3 chng c kh nng s dng du cao l MT3, M150, MX. Trong

chng M150 c kh nng s dng du mnh nht c ch s nh ho cao

72,7%. Chng ny c xc nh l Brevibacterium celere v c la

chn nghin cu tip

2.Xc nh c mt s cc c im sinh ho ca chng M150: c kh nng

kh NO3 thnh NO2, s dng arginin, ure, gluco iu kin hiu kh, s

dng yu gluconat, adipat, malat, khng c kh nng s dng, gelatin,

trytophan, escunin, caprat, ciprat v phenylacetat, gluco trong iu kin k

kh.

3.Xc nh c ng thi sinh tng hp CHHBMSH ca chng nghin cu

mnh nht t 96 gi n 144 gi vi ch s nh ho E24 t 72,8%, hot tnh

bt u gim i 168 gi ch cn 50%.

4.Xc nh c iu kin to CHHBM cao nht pH =7,5; nhit ph

hp l 30o C; nng NaCl thch hp l 0,5 %, ngun cacbon to CHHBM

mnh nht trong cc ngun cacbon nghin cu l du DO.5. Kh nng to CHHBM cao nht chng M150 l 11,6 g/l.

6.Xc nh c bn ca CHHBM theo nhit v thi gian: sau khi x l

nhit 1000 C hot tnh cn li 92,8% sau 30 pht v 78% sau 1 gi, cn

100% hot tnh sau 10 ngy gi 30oC ch gim cn 92,8% sau15 ngy gi,

55oC th hot tnh gim mnh hn sau 10 ngy cn 89%.

7. Kt qu th nghim cho thy CHHBM do chng M150 sinh ra c tc dng

tng cng kh nng phn hu du. Tc ng mnh khi b sung 2 n 3 ml.Kt lun CHHBMSH do chng ny sinh ra c kh nng ng dng x l mn

khoan.

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PH LC

Hnh 8. Kh nng nh ho ca chng M150 vi ccngun cacbon khc nhau

Hnh 9. Kh nng nh ho ca CHHBMSH ccnhit khc nhau

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Hnh 10. nh hng ca pH ti kh nng pht trin vnh ho du ca chng M150

Hnh 11. Kh nng nh ho ca chng M150 vicc nng mui khc nhau

Hnh 12. Hnh thi t bo ca chng M150

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Thu Hin, (2003), Nghin cu nh hng ca CHHBMSH to ra tchng vi khun Pseudomonas aeruginosa ASB ln tnh lu bin ca duth Bch H.

3. Li Thu Hin, (1997), Gio trnh cao hc vi sinh hc du m, Vin sinhthi v ti nguyn sinh vt, chung tm khoa hc t nhin v ti nguyn

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Nga, L Th Nhi Cng, Kiu Hu nh, (2003), Chn chng vi sinh vtto CHHBMSH cao ng dng trong cng nghip du kh v x l mitrng.

5. Tp ch thng tin du kh th gii s 7/ 2005.

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